U.S. patent number 5,340,926 [Application Number 08/102,697] was granted by the patent office on 1994-08-23 for process for the recovery of recombinantly produced protein from insoluble aggregate.
This patent grant is currently assigned to Celltech, Limited. Invention is credited to Sarojani Angal, Peter A. Lowe, Fiona A. O. Marston, Joyce A. Schoemaker.
United States Patent |
5,340,926 |
Lowe , et al. |
August 23, 1994 |
Process for the recovery of recombinantly produced protein from
insoluble aggregate
Abstract
In a process for the production of a soluble native protein,
such as immunoglobulin or methionine-prochymosin, in which an
insoluble form of the protein is produced by a host organism
transformed with a vector including a gene coding for the protein,
the insoluble form of the protein is reversibly denatured in an
alkaline aqueous solution at a pH selected to promote dissociation
of a group or groups of the protein involved in maintaining the
conformation of the protein, and the protein is subsequently
allowed to renature by reducing the pH of the solution below a pH
effective to denature the protein to produce the soluble native
form of the protein. The pH of the alkaline aqueous is suitably in
the range 9.0 to 11.5.
Inventors: |
Lowe; Peter A. (Reading,
GB), Marston; Fiona A. O. (Reading, GB),
Angal; Sarojani (Reading, GB), Schoemaker; Joyce
A. (London, GB) |
Assignee: |
Celltech, Limited
(GB)
|
Family
ID: |
27516505 |
Appl.
No.: |
08/102,697 |
Filed: |
August 5, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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726638 |
Jul 2, 1991 |
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411255 |
Sep 25, 1989 |
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676192 |
Nov 20, 1984 |
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Foreign Application Priority Data
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Mar 25, 1983 [GB] |
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8308234 |
Oct 12, 1983 [GB] |
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8327345 |
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Current U.S.
Class: |
530/423; 435/226;
435/252.3; 435/252.33; 435/69.1; 435/71.1; 435/71.2; 530/300;
530/305; 530/387.1; 530/412; 530/422 |
Current CPC
Class: |
C07K
1/1136 (20130101); C12N 9/6481 (20130101) |
Current International
Class: |
C07K
1/00 (20060101); C07K 1/113 (20060101); C12N
9/64 (20060101); C07K 003/12 (); C07K 015/06 () |
Field of
Search: |
;435/71.1,71.2,69.1,226,252.3,252.33
;530/300,305,387.1,412,422,423 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0068691 |
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Jan 1983 |
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EP |
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2100737 |
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Jan 1983 |
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GB |
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2138004 |
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Oct 1984 |
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GB |
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8304418 |
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Dec 1983 |
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WO |
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Other References
Zgirski et al. 1983 Acta Biochimica Bolonica 30: 33-38. .
Muluihill, D. et al., Chem Abst. vol. 87, No. 19, p. 216, Abst. No.
147755e, Nov. 7, 1977. .
Aknori, S. et al., Chem. Abst., vol. 97, No. 19, p. 528, Abst No.
1608005, Nov. 8, 1982. .
Wetzel, R. et al., Gene, vol. 16, pp. 63-71, 1981. .
Wetzel, R. et al., Biochemistry, vol. 19 pp. 6096-6104, 1980. .
Freedman, R. et al., The Enzymology of Post Transcriptional
Modification of Proteins, vol. 1, pp. 166-170, 1980. .
Mahler, M. et al Biological Chemistry (2nd Ed) Harper & Row,
New York, N.Y., 177-183, 1971..
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Primary Examiner: Schwartz; Richard A.
Assistant Examiner: LeGuyader; J.
Attorney, Agent or Firm: Woodcock Washburn Kurtz Mackiewicz
& Norris
Parent Case Text
This is a continuation of application Ser. No. 07/726,638, filed
Jul. 2, 1991, now abandoned, which is a continuation of Ser. No.
07/411,255, filed Sep. 25, 1989, now abandoned; which is a
continuation of application Ser. No. 06/676,192 filed Nov. 20,
1984, now abandoned, which is the U.S. National application of PCT
application PCT/GB84/00093 filed Mar. 23, 1984.
Claims
We claim:
1. In a process for the production of a soluble native protein
which comprises the steps:
(a) culturing a host organism transformed with a vector including a
gene coding for said protein to produce an insoluble aggregate form
of said protein;
(b) reversibly denaturing said insoluble aggregate form of said
protein by adding said insoluble aggregate form of said protein to
a first solution comprising an aqueous solution of urea or
guanidine hydrochloride, and
(c) subsequently renaturing the protein whereby the soluble native
form of said protein is produced,
the improvement of enhancing the yield of native protein wherein a
second solution is subsequently added to step (b) comprising an
aqueous solution of an alkali metal hydroxide or an alkali metal
extraction buffer, which second solution and the combined first and
second solutions having a pH which dissociates groups of said
protein which maintain conformation of said protein, and reducing
in step (c) the pH of the combined aqueous solutions of step (b) to
a level below that effective to denature the protein.
2. The process of claim 1 wherein the second solution of step b is
an aqueous solution of an alkali metal hydroxide.
3. The process of claim 2 wherein the alkali metal hydroxide is
sodium hydroxide.
4. The process of claim 2 wherein the alkali metal hydroxide is
potassium hydroxide.
5. The process according to claim 2 wherein the volume of said
second solution is from 10 to 50 times greater than the volume of
said first solution, wherein said second solution has a pH from 10
to 11.5, and wherein said alkali metal hydroxide is sodium
hydroxide or potassium hydroxide.
6. The process according to claim 5 wherein the first solution
comprises an aqueous solution of urea at a concentration of at
least 7M.
7. The process according to claim 5 wherein the first solution
comprises an aqueous solution of guanidine hydrochloride at a
concentration of at least 6M.
8. The process of claim 1 wherein the second solution of step b is
an aqueous solution of the alkali extraction buffer K.sub.2
HPO.sub.4.
9. The process of claim 1 wherein the second solution of step b is
an aqueous solution of the alkali extraction buffer KH.sub.2
PO.sub.4.
10. The process according to claim 1 wherein said second solution
has a pH from 9 to 11.5.
11. The process according to claim 1 wherein the insoluble form of
the protein is present in conjunction with debris derived from the
host organism, said debris being insoluble in said second
solution.
12. The process of claim 1 wherein the volume of said second
solution added is from 10 to 50 times greater than the volume of
the first solution.
13. The process of claim 1 wherein the first solution comprises an
aqueous solution of urea at a concentration of at least 7M.
14. The process of claim 1 wherein the first solution comprises an
aqueous solution of guanidine hydrochloride at a concentration of
at least 6M.
Description
FIELD OF THE INVENTION
This invention relates to the field of protein production using
recombinant DNA biotechnology. In particular it relates to a
process for the recovery of a protein produced in an insoluble form
by a host organism transformed with a vector including a gene
coding for the protein.
BACKGROUND OF THE INVENTION
There are now numerous examples of commercially valuable proteins
which may be produced in large quantities by culturing a host
organism capable of expressing heterologous genetic material. Once
a protein has been produced by a host organism it is usually
necessary to treat the host organism in some way, in order to
obtain the desired protein. In some cases, such as in the
production of interferon in Escherichia coli a lysis or
permeabilisation treatment alone may be sufficient to afford
satisfactory yields. However, some proteins are produced within a
host organism in the form of insoluble protein aggregates which are
not susceptible to extraction by lysis or permeabilisation
treatment alone. It has been reported that a human insulin fusion
protein produced in E. coli forms insoluble protein aggregates (see
D. C. Williams et al (1982) Science 215 687-689).
A protein exists as a chain of amino acids linked by peptide bonds.
In the normal biologically active form of a protein (hereinafter
referred to as the native form) the chain is folded into a
thermodynamically preferred three dimensional structure, the
conformation of which is maintained by relatively weak interatomic
forces such as hydrogen bonding, hydrophobic interactions and
charge interactions. Covalent bonds between sulphur atoms may form
intramolecular disulphide bridges in the polypeptide chain, as well
as intermolecular disulphide bridges between separate polypeptide
chains of multisubunit proteins, e.g. insulin. The insoluble
proteins produced in some instances do not exhibit the functional
activity of their natural counterparts and are therefore in general
of little use as commercial products. The lack of functional
activity may be due to a number of factors but it is likely that
such proteins produced by transformed host organisms are formed in
a conformation which differs from that of their native form. They
may also possess unwanted intermolecular disulphide bonds not
required for functional activity of the native protein in addition
to intramolecular disulphide bonds. The altered three dimensional
structure of such proteins not only leads to insolubility but also
diminishes or abolishes the biological activity of the protein. It
is not possible to predict whether a given protein expressed by a
given host organism will be soluble or insoluble.
In our copending British Patent Application GB2100737A (an
identical disclosure of which is contained in assignee's U.S.
application Ser. No. 389,063, filed Jun. 16, 1982 now abandoned) we
describe a process for the production of the proteolytic enzyme
chymosin. The process involves cleaving a chymosin precursor
protein produced by a host organism which has been transformed with
a vector including a gene coding for the relevant protein. In the
course of our work we discovered that the chymosin precursor
proteins were not produced in their native form but as an insoluble
aggregate. In order to produce a chymosin precursor in a native
form which may be cleaved to form active native chymosin, the
proteins produced by a host organism were solubilised and converted
into their native form before the standard techniques of protein
purification and cleavage could be applied.
In our copending published International Patent Application WO
83/04418 the methods used for the solubilisation of chymosin
precursor proteins are described. In general the techniques
described involve the denaturation of the protein followed by the
removal of the denaturant thereby allowing renaturation of the
protein. In one example the denaturant used is a compound such as
urea or guanidine hydrochloride. When the insoluble precursor is
treated with urea or guanidine hydrochloride it is solubilised.
When the denaturant is removed, for example by dialysis, the
protein returns to a thermodynamically stable conformation which,
in the case of chymosin precursors, is a conformation capable of
being converted to active chymosin.
The solubilised protein may be separated from insoluble cellular
debris by centrifugation or filtration. The production of proteins
from suitably transformed host organisms is potentially of great
commercial value. The processes involved are of a type which may be
scaled up from a laboratory scale to an industrial scale. However,
where the protein produced is formed as an insoluble aggregate,
potential complications in the process may increase the cost of
production beyond a viable level. The solubilisation technique
described above, whilst effective to solubilise such proteins, is
relatively expensive and may represent a significant production
cost.
We have discovered a generally applicable solubilisation process
which, in its broadest aspect, does away with the requirement of
relatively expensive reagents.
SUMMARY OF THE INVENTION
According to the present invention we provide a process for the
production of a soluble native protein in which an insoluble form
of the protein is produced by a host organism transformed with a
vector including a gene coding for the protein, wherein the
insoluble form of the protein is reversibly denatured in an
alkaline aqueous solution at a pH selected to promote dissociation
of a group or groups of the protein involved in maintaining the
conformation of the protein and the protein is subsequently allowed
to renature by reducing the pH of the solution below a pH effective
to denature the protein to produce the soluble native form of the
protein.
The use of an alkali solution to denature the insoluble protein
reduces the reagent cost of the process. The pH is selected with
reference to the protein to which the process is to be applied. In
particular the pH is selected such that groups responsible for
holding the protein in an unnatural conformation by means of
intramolecular, or potentially in the case of a protein aggregate
nonfunctional intermolecular, bonds or forces are dissociated such
that, when the pH is reduced, the protein refolds in the native
conformation. The groups responsible for holding the protein in an
unnatural conformation may be ionisable groups, in which case the
pH is preferably selected to be compatible with the pKa of the
relevant ionizable group.
Studies in our laboratory have shown that intermolecular disulphide
bonds exist in prochymosin aggregates produced in E. coli
(Schoemaker et al (1984) submitted to PNAS). Native prochymosin is
monomeric and contains three intramolecular disulphide bonds
(Foltmann et al (1977) Proc. Natl. Acad. Sci. U.S.A. 74 pp
2321-2324). Six thiol groups per molecule are therefore available
to form intermolecular and intramolecular bonds within the protein
aggregate. Consequently, disulphide bonds must be broken and
correctly reformed for denaturation/renaturation to successfully
solubilise prochymosin. This may be achieved by using an alkaline
aqueous solution of pH 10.7 (.+-.0.5). The free thiol groups of
cysteine have a pKa value of 10.46.
The term "insoluble" as used herein means in a form which, under
substantially neutral conditions (for example pH in the range 5.5
to 8.5), is substantially insoluble or is in an insolubilised
association with insoluble material produced on lysis of host
organism cells. The insoluble product is either produced within the
cells of the host organism in the form of insoluble relatively high
molecular weight aggregates or may simply be associated with
insoluble cell membrane material. The process permits the
separation of solubilised protein from insoluble cellular
debris.
Any suitable alkali may be used in the process, for example an
aqueous solution of an alkali metal hydroxide such as NaOH or KOH,
an aqueous buffer, or an aqueous solution of an organic base such
as triethylamine.
Preferably the alkaline aqueous solution has a pH of from 9 to
11.5, most preferably from 10 to 11.
The treatment of an insoluble protein with an alkaline aqueous
solution may not, in all cases, result in complete solubilisation
of the protein. Since insoluble material is present at all times, a
number of mass transfer effects may be important. It has been found
that multiple extractions with alkali are more efficient than a
single extraction even when large extraction volumes are used. This
also has the advantage of minimising the time for which the
solubilised protein is in contact with alkali. Preferably,
therefore, one or more extractions of denatured protein are
performed.
The methods of solubilisation in a strong denaturant such as
guanidine hydrochloride or urea described in published British
patent application GB2100737A and in published International patent
application WO 83/04418 and the methods of solubilisation using
alkali, according to the broad aspect of the present invention each
solubilise significant percentages of insoluble proteins found in
extracts from host organisms. However, neither is completely
quantitative in terms of recovery of native protein. The reasons
for this have not been clearly defined and are probably different
for the two types of solubilisation. It appears that guanidine
hydrochloride solubilises all the material present but only a
portion is converted into native proteins after removal of
guanidine hydrochloride. Alkali treatment may not allow complete
renaturation to form the native form of the protein and in addition
does not solubilise all of the insoluble form of the protein. We
have discovered that by combining the two methods a greatly
enhanced yield of native protein may be obtained.
According to a preferred aspect of the invention the insoluble form
of the protein is first denatured in an aqueous solution, and
subsequently the resulting solution is diluted in an alkaline
aqueous solution at a pH selected to promote dissociation of the
group or groups of the protein involved in maintaining the
conformation of the protein and the protein is renatured by
reducing the pH of the solution below a pH effective to denature
the protein, to produce the soluble native form of the protein.
The dilution introduces an element of physical separation between
the denatured molecules, before renaturation is brought about, for
example, by neutralisation of the alkaline denaturing solution. The
dilution and resulting physical separation of the denatured
molecules appears to assist their renaturation in native form. The
solubilisation process described immediately above leads to a
recovery, in the case of methionine-prochymosin, of more than 30%
compared to, for example, 10 to 20% for the multiple alkali
extractions also described above.
Preferably the pH of the alkaline aqueous solution is from 9 to
11.5, most preferably from 10 to 11.
Preferably the dilution is from 10 fold to 50 fold. (That is a
dilution into a total volume of from 10 to 50 volumes).
Preferably, in the combined solubilisation process described above
the insoluble protein is denatured in an aqueous solution
comprising urea at a concentration of at least 7M or in a solution
comprising guanidine hydrochloride at a concentration of at least
6M.
The insoluble protein may be a recombinant animal protein produced
by a host organism. Examples of such proteins are immunoglobin
light and heavy chain polypeptides, foot and mouth disease antigens
and thymosin and insulin proteins.
The host organism may be a naturally occuring organism or a mutated
organism capable of producing an insoluble protein. Preferably,
however, the host organism is an organism or the progeny of an
organism which has been transformed using recombinant DNA
techniques with a heterologous DNA sequence which codes for the
production of a protein heterologous to the host organism and which
is produced in an insoluble form. The host organism may be a
eukaryotic organism such as a yeast or animal or plant cell.
Preferred yeasts include Saccharomyces cerevisiae and
kluyveromyces. In the alternative the host organism may be a
bacterium such as E. coli, B. subtilis, B. stearothermophilis or
Pseudomonas. Examples of specific host organism strains include E.
coli HB101, E.coli X1776, E.coli X2882, E.coli PS410, E. coli RV308
and E.coli MRC1.
The host organism may be transformed with any suitable vector
molecule including plasmids such as colE1, pCR1, pBR322, RP4 and
phage .lambda. DNA or derivatives of any of these.
Prior to treatment with the process of the present invention the
host cells may be subjected to an appropriate lysis or
permeabilisation treatment to facilitate recovery of the product.
For example, the host organism may be treated with an enzyme, for
example a lysozome, or a mechanical cell destructing device to
break down the cells.
The process of the invention may then be employed to solubilise the
insolubilised product and the resulting solution may be separated
from solid cell material such as insoluble cell membrane debris.
Any suitable method including filtration or centrifugation may be
used to separate solution containing the solubilised protein from
the solid cell material.
The present invention is now illustrated by way of the following
Examples:
EXAMPLE 1
An experiment was conducted in which the solubilisation of
insoluble methionine-prochymosin produced by E. coli cells
transformed with vector pCT70 was achieved using alkaline
denaturation. The preparation of the transformed E.coli cell line
is described in detail in published British patent application
GB2100737A.
Frozen E.coli/pCT 70 cells grown under induced conditions were
suspended in three times their own weight of of 0.05M Tris-HCl pH
8, 1 mM EDTA, 0.1M NaCl, containing 23 .mu.g/ml
phenylmethylsulphonylfluoride (PMSF) and 130 .mu.g/ml of lysozyme
and the suspension was incubated at 4.degree. C. for 20 minutes.
Sodium deoxycholate was added to a final concentration of 0.5% and
10 ug of DNA ase 1 (from bovine pancreas) was added per gram of
E.coli starting material. The solution was incubated at 15.degree.
C. for 30 minutes by which time the viscosity of the solution had
decreased markedly. The extract, obtained as described above, was
centrifuged for 45 minutes at 4.degree. C. and 10000.times.g. At
this stage effectively all the methionine-prochymosin product was
in the pellet fraction in insolubilised form, presumably as a
result of aggregation or binding to cellular debris. The pellet was
washed in 3 volumes of 0.01M tris-HCl. pH8, 0.1 M NaCl, 1 mM EDTA
at 4.degree. C. After further centrifugation, as above, the
supernatant solution was discarded and the pellet resuspended in 3
volumes of alkali extraction buffer: 0.05M K.sub.2 HPO.sub.4, 1 mM
EDTA, 0.1M NaCl, pH 10.7 and the suspension adjusted to pH 10.7
with sodium hydroxide. The suspension was allowed to stand for at
least 1 hour (and up to 16 hours) at 4.degree. C., the pH of the
supernatant adjusted to 8.0 by addition of concentrated HCl and
centrifuged as above. Methionine-prochymosin, representing a
substantial proportion of the methionine-prochymosin originally
present in the pellet, was found to be present in the supernatant
in a soluble form which could be converted to catalytically active
chymosin by acidification/neutralisation activation treatment
substantially as described in published British patent application
GB2100737A.
We further noted that re-extraction of the debris left after the
first alkali extraction liberates an equivalent amount of
prochymosin. Alkali extraction may be repeated to a total of 4-5
times with the liberation of approximately equivalent levels of
proychmosin at each extraction.
EXAMPLE 2
An experiment was conducted in which the solubilisation of an
insoluble immunoglobulin light chain polypetptide produced by
E.coli cells transformed with vector pNP3 was achieved using
alkaline denaturation. The preparation of the transformed E.coli
cell line is described in copending international patent
application No. PCT/GB 84/00094, field March, 1984 (the U.S.
national application of which is Ser. No. 06/672,265, filed Nov.
14, 1984, now U.S. Pat. No. 4,816,397) of even date herewith.
E.coli cells transformed with the plasmid pNP3, containing a gene
coding for the .lambda..sub.1 light chain of the
4-hydroxy-3-nitrophenyl acetyl (NP) binding monoclonal antibody
S43, were grown under inducing conditions. The cells were harvested
and resuspended in 0.05M TRIS pH 8.0, 0.233M NaCl. 5% glycerol v/v
containing 130 .mu.g/ml of lysozyme and incubated at 4.degree. C.
or room temperature for 20 minutes. Sodium deoxycholate was then
added to a final concentration of 0.05% and 10 .mu.g of DNA ase 1
(from bovine pancreas) was added per gm wet wt of E.coli. The
solution was then incubated at 15.degree. C. for 30 minutes by
which time the viscosity of the solution had decreased markedly.
The resultant mixture was then centrifuged (at 10,000.times.g for
15 minutes for small volumes (1 ml) or 1 hour for larger volumes).
Immunoprecipitation studies indicated that the .lambda. light chain
protein was present in the insoluble fraction rather than the
soluble fraction.
In order to purify the recombinant light chain, the E.coli pellet
fraction, obtained as described above, was resuspended in a pH 11.5
buffer comprising 50 mM K.sub.2 HPO.sub.4, 0.1M NaCl and 1 mM EDTA.
The suspension was allowed to stand for at least 1 hour (and up to
16 hours), centrifuged as above and the pH of the supernatant
adjusted to 8.0 by addition of concentrated HCl. A substantial
proportion of the .lambda. light chain protein, originally present
in the pellet was found to be present in the supernatant is a
soluble form.
EXAMPLE 3
An experiment was conducted in which the solubilisation of
methionine-prochymosin produced by E.coli cells transformed with
vector pCT70 was achieved using denaturation with guanidine
hydrochloride, followed by dilution into an alkaline solution. The
preparation of the transformed cell line is described in detail in
published British patent application GB2100737A.
E.coli/pCT 70 cell debris containing insoluble
methionine-prochymosin was prepared and washed as described in
Example 1 above and the following manipulations were carried out at
room temperature. The cell debris was dissolved in 3-5 volumes of
buffer to final concentration of 6M guanidine HCl/0.05M Tris pH8, 1
mM EDTA, 0.1 m NaCl and allowed to stand for 30 minutes-2 hours.
The mixture was diluted into 10-50 volumes of the above buffer at
pH 10.7 lacking guanidine HCl. Dilution was effected by slow
addition of the sample to the stirred diluent over a period of
10-30 minutes. The diluted mixture was readjusted to pH 10.7 by the
addition of 1M NaOH and allowed to stand for 10 minutes-2 hours.
The pH was then adjusted to 8 by the addition of 1N HCl and the
mixture allowed to stand for a further 30 minutes before
centrifuging as above to remove precipitated proteins. The
supernatant so produced contained soluble methionine-prochymosin
which could be converted to catalytically active chymosin by
acidification and neutralisation and purified as described in
published British patent application GB2100737A. In a very similar
experiment an 8M urea buffer was used in place of the 6M guanidine
HCl buffer described above. The results were as described
above.
EXAMPLE 4
An experiment was conducted in which the solubilisation of
methionine-prochymosin produced by E.coli cells transformed with
vector pCT 70 was achieved using a method similar to that described
in Example 3. The preparation of the transformed cell line is
described in detail in published British patent application
GB2100737A.
E.coli/pCT 70 cell debris containing insoluble
methionine-prochymosin was prepared and washed as described in
Example 1 above and the following manipulations were carried out at
room temperature. The washed pellets were suspended in a buffer
containing 50 mM Tris HCl pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.1 mM
PMSF supplemented with 8M urea (deionized). For every 10 g weight
of starting material, 90 ml of buffer were used. After 1 hour, this
solution was added slowly to a buffer containing 50 mM KH.sub.2
PO.sub.4 pH 10.7 containing 1 mM EDTA and 50 mM NaCl and left for
at least 30 minutes. Various dilutions were made in order to
establish an optimum dilution range. This proved to be a dilution
in alkali of between 10 and 50 fold. The pH was maintained at pH
10.7 for the period and then adjusted to pH 8. The product was
activated to give active chymosin and the level of chymosin
activity was assayed (Emtage, J. S., Angal S., Doel, M. T., Harris,
T. J. R., Jenkins, B., Lilley, G., and Lowe, P. A. (1983) Proc.
Natl. Acad. Sci. USA 80, 3671-3675). The results are shown in Table
1.
TABLE 1 ______________________________________ Effect of dilution
on the recovery of milk clotting activity. MILK FOLD DILUTION
CLOTTING ACTIVITY UREA ALKALI OVERALL (mg)
______________________________________ 1 10 10 0.04 1 25 25 0.10 1
50 50 0.05 5 10 50 0.10 5 25 125 0.09 5 50 250 1.23 10 10 100 1.97
10 25 250 1.56 10 50 500 0.09
______________________________________
EXAMPLE 5
An experiment was conducted in which the solubilisation of
insoluble immunoglobulin heavy and light chains produced together
in E. coli was achieved using denaturation with urea followed by
dilution into alkali. The preparation of the transformed cell line
is described in copending international patent application No.
PCT/GB 84 of even data herewith.
In order to produce functional antibodies from E. coli cells
expressing the genes for both the heavy and light immunoglobulin
chains, the cells were lysed, the insoluble material was washed
followed by sonication (three times for 3 minutes). The material
was then dissolved in 9M urea 50 mm Glycine-Na+ pH10.8 1 Mm EDTA
and 20 mM 2-mercaptoethanol. This extract was dialysed for 40 hours
against three changes of 20 vols. of 100 mM ECl 50 Mm Glycine-Na+
pH10.8 5% glycerol 0.5 mM EDTA 0.5 mM reduced glutathione and 0.1
mM oxidised glutathione. The dialysate was cleared by
centrifugation at 30,000 g for 15 minutes and loaded directly onto
DEAE sephacel followed by development with 0-0.5M KCl linear
gradient in 10 mM Tris-HCl 0.5 mM EDTA pH8.0.
* * * * *